1. Fly rearing and collection

1. Maintain flies at 25 °C in narrow vials containing standard Drosophila food.
   NOTE: The sample size for each genotype should comprise at least nine replicates, each consisting of a single respirometer chamber containing 15-25 flies, set up as described below.
2. Transfer the flies every 2-3 days.
3. Anesthetize flies with CO₂, collect groups of 15-25 males of each genotype, and place each group into fresh, unyeasted food vials.
   NOTE: Males were used to reduce variability due to reproductive status. The method applies to both sexes.
4. Allow the flies to recover at 25 °C for at least 24 h.
   NOTE: By the time of the experiment, flies should be 1-4 days old. The frequency of collections described in step 1.3 can be set to narrow the age range of the flies.

2. Setup and assembly of respirometer chamber

1. Turn on the water bath and set it to the desired temperature for the experiment.
   NOTE: The experiments below were conducted at 25 °C using 50 mL Schlenk tubes as chambers. Components are to be assembled as shown in Figures 1A, 1B, and 1C.
2. Clean the ground glass joints of chambers and sensor plugs thoroughly by spraying 70% ethanol onto a laboratory wipe (not directly onto the joint) and wiping dust and old grease from the sensor plug (Figure 1A). Wipe off ethanol with a fresh laboratory wipe.
3. Place 1 cm piece of cotton roll soaked in purified water into the bottom of the chamber to stabilize the humidity.
   1. Add enough water (~0.5 mL) to form a small pool at the bottom of the cotton roll.
4. Wipe off any water that has spilled onto the joint of the chamber.
5. Transfer the flies to labeled polypropylene tubes using a funnel.
   1. Plug the tube with a cotton roll.
      NOTE: Tubes consist of a 5 mL polypropylene test tube, trimmed to 5.5 cm in length and perforated with a hot knife to allow the free exchange of air with the experimental chamber. CO₂ anesthesia is known to cause metabolic abnormalities, so flies are transferred without anesthesia which requires more care to avoid losing the flies.
6. Add one ventilated tube with flies into each respirometer chamber (on top of wet cotton).
7. Fill soda lime cartridges (4-5 pellets per tube) and place them on the top of the tube containing flies inside the chamber.
   NOTE: Soda lime cartridges consist of 800 µL centrifuge tubes perforated 4-5 times with a power drill.
8. Fill O₂ generators with saturated copper sulfate (CuSO₄) solution below level of vent holes
   NOTE: O₂ generators consist of screw-cap centrifuge tubes with 4 holes drilled below the threads. Platinum (Pt) and Copper (Cu) electrodes are soldered to two-pin connector, inserted into holes drilled in cap, and affixed with epoxy. Electrolysis of CuSO₄ generates the O₂ consumed by the experimental organism. CuSO₄ is toxic to invertebrates, avoid spills or leakage and clean up immediately.
9. Connect the filled O₂ generator to two-pin connector on the sensor plug.
   NOTE: The copper cathode must connect with the negative output of the controller and the platinum anode to the positive wire. Reversed connections will cause the failure of the experiment.
10. Place two small dabs of clear silicone grease on opposite sides of the ground glass joint of the sensor plug.
11. Insert the plug into the chamber and rotate the plug (or chamber) with moderate pressure to spread the grease in the joint.
    1. Wipe off excess grease with a laboratory wipe.
12. Snap plastic Keck clamps onto joints to secure plugs in chambers. The assembled chamber should look like Figure 1C.
13. Repeat the above steps for the number of chambers used for the day's experiment.
    NOTE: The number of chambers that can be recorded is limited by the number of available chambers, controllers and USB inputs to the computer. For the present experiments, seven chambers were normally run in parallel. Experimental flies such as mutants should be matched with appropriate controls. A chamber set up identically but without flies should be included in each experiment as a control for environmental variation. Chambers containing different treatments (mutant, wildtype, no-fly) should be rotated between experiments.
14. Place assembled chambers into a rack in the water bath with stopcocks open (Figure 1E).
    NOTE: To avoid circadian variation, chambers were placed into the bath between 9:30 and 9:50 am for all experiments described here.
15. Leave stopcocks open (Keep the handle parallel to the stopcock).
    NOTE: Be careful not to allow water to enter the stopcocks.
16. Allow the chambers to equilibrate with stopcocks open for about 30 min.
    ​NOTE: While the chamber is equilibrated, connect the electronics and set up data acquisition as described below.

3. Setting up controllers and computer

1. Be sure that the switches supplying current to the O₂ generators are in the OFF position (away from the connector; Figure 1D).
2. Plug each controller box into an available Universal serial bus (USB) port.
   NOTE: Construction and programming of controller units described elsewhere¹².
3. Connect controllers to respirometer chambers using 6-conductor cables.
4. Check that the organic light emitting diode (OLED) displays of the controllers (Figure 1D) are displaying environmental parameters.
5. Briefly turn on O₂ generators using the switch on the controller (Figure 1D).
   1. If the current value increases from zero to between 35 and 55 mA, the controller and chamber are ready for experiments.
6. Determine which COM ports are being used by the controllers, as described below.,
   1. Click the Start Icon in Microsoft Windows.
   2. Click the Settings Icon.
   3. Click Bluetooth and Devices.
   4. Ensure that the controllers and their COM ports appear in the list of devices.
7. Open PuTTY on the desktop and set up a log file for each channel of the respirometer as described below.
   NOTE: PuTTY is a free secure shell and telnet client that is used to transfer data to the computer via COM ports.
   1. Select COM port for a controller by typing the number of the port in the "Serial line" box (Figure 2A).
   2. Click on Logging.
   3. Select Printable Output in "Session logging" (Figure 2B).
   4. Under Log File Name click Durchsuchen.
   5. In the folder of the choice, create a filename containing descriptive information (e.g., date, species, COM port number).
   6. Click Save.
   7. Click Open. A window will open showing comma-delimited data being logged (Figure 2C).
   8. Repeat for all other controllers in use for the experiment. Input to each COM port will appear as a separate window (Figure 2D).

4. Running experiments

1. Once chambers have equilibrated for 30 min, seal them by closing stopcocks.
2. Cover the bath and chambers with a polystyrene foam box to maintain a stable environment.
3. Allow to equilibrate for another hour.
4. Turn on the current to the O₂ generator of each chamber using the switch on the controller box.
5. Once the O₂ generators are activated, ensure that the pressure increases to pre-set OFF pressure.
   NOTE: 1017 hPa, which is slightly above atmospheric pressure, was used as the "OFF" pressure in this series of experiments. Return to the ambient pressure will indicate leakage of gas from the chambers. Further, it allows the same pressure to be used across experiments regardless of ambient barometric pressure. The "ON" pressure was 1016 hPa, meaning that pressure only needed to drop 1 hPa before the O₂ generator was activated. This provided adequate sensitivity to measure O₂ consumption in Drosophila. Once a chamber is pressurized to the "OFF" setting, current should drop to zero.
6. Let the experiment run for 3 or more h.
   NOTE: Higher V_O2 at elevated temperatures can allow for shorter experiment times. Monitor occasionally to ensure that equipment is functioning but avoid excessive activity near the chambers that may affect temperature stability.

5. Finishing experiment

1. Turn off O₂ generators on all controllers.
   NOTE: Do first to avoid running the O₂ generators while the chambers are open.
2. Open the stopcocks to unseal the chambers.
3. Leave the PuTTY windows open for another 5-15 min to provide a final baseline.
4. Close the PuTTY window for each controller, ending recordings.
   ​NOTE: All experiments ended between 4:50 and 5:10 pm.
5. Disconnect sensors from cables.
6. Move chambers to dry rack.
7. Remove sensor plugs one at a time from the chambers.
8. Disconnect the O₂ generators and place them in the tube rack.
9. Wipe grease off the sensor plug and keep it in the rack.
10. Clean grease from chamber joints and remove tubes with flies and soda lime.
11. Anesthetize flies in each tube with CO_2, tap onto a weight boat and weigh on a microbalance.
    1. Log the weight and number of flies for each tube.
12. Discard flies or set them aside for additional procedures.
13. Dump soda lime from cartridges into the waste container.
14. Open the O₂ generator and discard the CuSO₄ solution into the waste container.
    1. Rinse electrodes and tube with purified water.
    2. Place the tube racks for drying.

6. Analysis of charge transfer data

1. Import Data as comma-delimited text into a spreadsheet, with each record comprising a separate worksheet.
2. Record the current and time data for each pulse of the O₂ generator. Starting with the first pulse after the chamber was pressurized, record the start time and end time (as row numbers) of each current pulse. That is the row number when the current goes above zero (usually to about 45-50 mA) to the last row that is above zero.
3. Make a table on the worksheet to record the following data:
   1. The average current amplitude during the pulse: = AVERAGE([first row of pulse]:[last row of pulse]) for each pulse (from the current column).
   2. Pulse duration: ([Last row of pulse] – [first row of pulse[-one row]])/1000 for each pulse (from the time in milliseconds column).
   3. Total experiment time: [time at start of last pulse] – [time at end of first pulse after chamber pressurized] (from the time in minutes column).
4. Then calculate charge transfer (Q) for each pulse (average current X duration)
5. Sum the charge from all pulses to calculate Total Charge (Q_tot).

7. Analysis of O₂ consumption

1. Set up a new spreadsheet for all data and enter or calculate the following for each chamber:
   1. Q_tot (total charge)
   2. Moles (= Q ÷ 96485 × 4)
   3. mL O₂ (= moles × 22413 mL/mol)
   4. Total time (from the data analysis above)
   5. mL min^-1 (= ml O₂ ÷ total time)
   6. Weight in grams (flies anesthetized and weighed measured after the experiment)
   7. mL min^-1 g^-1 (= mL min^-1 ÷ weight in grams)
   8. mL/h/g (the above × 60)
   9. mg/fly (= weight of flies ÷ number of flies)
   10. μL fly^-1 h^-1 (= (mL min^-1 × 3600) ÷ number of flies).
2. Tabulate data for each treatment (genotype, e.g.)
3. Compare treatments using ANOVA, t-test, or Mann-Whitney u-test ¹³.